1. Field of the invention
The present invention relates generally to compounds and methods for identifying compounds which have a biological activity. More particularly, the present invention relates to methods for identifying compounds capable of inhibiting the production of .beta.-amyloid peptide in cells.
Alzheimer's disease is a neurodegenerative brain disorder which typically leads to progressive memory loss, dementia, and finally death. It is the fourth leading cause of death in the United States, responsible for approximately 100,000 deaths annually. Alzheimer's disease primarily afflicts the elderly and is expected to be an increasing health care concern in the future with the aging of the United States population. At present, there are no proven means for diagnosis, prevention, treatment, or cure of Alzheimer's disease.
Alzheimer's disease is characterized by the presence of numerous amyloid plaques and neurofibrillary tangles (highly insoluble protein aggregates) present in the brains of Alzheimer's disease patients, particularly in those regions involved with memory and cognition. While in the past there was significant scientific debate over whether the plaques and tangles are a cause or are merely the result of Alzheimer's disease, recent discoveries indicate that amyloid plaque is a causative precursor or factor. In particular, it has been discovered that the production of .beta.-amyloid peptides, a major constituent of the amyloid plaque, can result from mutation in the gene encoding amyloid precursor protein, a protein which when normally processed will not produce the .beta.-amyloid peptide. Moreover, .beta.-amyloid peptide is toxic to brain neurons, and neuronal cell death is associated with the disease.
Thus, it is believed that a drug that could interfere with .beta.-amyloid plaque formation or toxicity may delay or halt the progression of Alzheimer's disease. At present, no suitable in vitro systems or methods exist for screening candidate drugs for the ability to inhibit or prevent the production of .beta.-amyloid plaque. The scarcity of such screening methods may, at least in part, result from insufficient understanding of the pathogenic mechanism(s) which cause the conversion of amyloid precursor protein to the .beta.-amyloid peptide, and ultimately to the amyloid plaque.
For these reasons, it would be desirable to provide methods and systems for screening test compounds for the ability to inhibit or prevent the conversion of amyloid precursor protein to .beta.-amyloid peptide. In particular, it would be desirable to base such methods and systems on a metabolic pathway which is involved in such conversion, where the test compound would be able to interrupt or interfere with the metabolic pathway which leads to conversion. Such methods and systems should be rapid, economical, and suitable for screening large numbers of test compounds. In particular, initial methods should utilize in vitro systems rather than animal models, so that the methods are particularly suitable for initial screening of test compounds to identify suitable candidate drugs.
2. Description of the Background Art
.beta.-amyloid peptide (also referred to as A4 or A.beta.P) is derived from amyloid precursor protein, which is expressed in differently spliced forms of 695, 751, and 770 amino acids. See, Kang et al. (1987) Nature 325:773-776; Ponte et al. (1988) Nature 331:525-527; and Kitaguchi et al. (1988) Nature 331:530-532. Normal processing of amyloid precursor protein involves proteolytic cleavage at a site between residues 16 and 17 (as numbered starting with Asp.sub.597 as residue 1) near the transmembrane domain, resulting in the constitutive secretion of an extracellular domain and the appearance of an intracellular fragment (approximately 9 kilodalton (kD)), referred to as the constitutive carboxy-terminal fragment (cCTF). This pathway appears to be widely conserved among species and present in many cell types. See, Weidemann et al. (1989) Cell 57:115-126; Oltersdorf et al. (1990) J. Biol. Chem. 265:4492-4497; and Esch et al. (1990) Science 248:1122-1124. This normal pathway cleaves within the region of the precursor protein which corresponds to the .beta.-amyloid peptide, thus apparently precluding its formation.
Tamaoka et al. (1991) Am. Assoc. Neuropathol. Abstr. 149, report the identification of an approximately 22 kD fragment of amyloid precursor protein microvessel fractions from human cerebral cortex labelled by four distinct antisera to amyloid precursor protein C-terminal peptides as well as by antibodies raised to fusion proteins containing residues 444-592 (.alpha.BX5) and residues 595-695 (.alpha.BX6) of amyloid precursor protein.
Golde et al. (1991) Soc. Neuroscience Abstr. 17:1293, identify a set of carboxy-terminal fragments of amyloid precursor protein ranging in molecular weights from 8 to 12 kD, the largest of which the authors believe may contain the entire .beta.-amyloid peptide sequence and which is produced in the normal processing of amyloid precursor protein in 293 cells (a human kidney cell line) overexpressing amyloid precursor protein.